Development of Tailored Graphene Nanoparticles: Preparation, Sorting and Structure Assessment by Complementary Techniques.

We present a thorough structural characterization of Graphene Nano Particles (GNPs) prepared by means of physical procedures, i.e., ball milling and ultra-sonication of high-purity synthetic graphite. UV-vis absorption/extinction spectroscopy, Dynamic Light Scattering, Transmission Electron Microscopy, IR and Raman spectroscopies were performed. Particles with small size were obtained, with an average lateral size = 70−120 nm, formed by few = 1−10 stacked layers, and with a small number of carboxylic groups on the edges. GNPs relatively more functionalized were separated by centrifugation, which formed stable water dispersions without the need for any surfactant. A critical reading and unified interpretation of a wide set of spectroscopic data was provided, which demonstrated the potential of Specular Reflectance Infrared Spectroscopy for the diagnosis and quantification of chemical functionalization of GNPs. Raman parameters commonly adopted for the characterization of graphitic materials do not always follow a monotonic trend, e.g., with the particle size and shape, thus unveiling some limitations of the available spectroscopic metrics. This issue was overcome thanks to a comparative spectra analysis, including spectra deconvolution by means of curve fitting procedures, experiments on reference materials and the exploitation of complementary characterization techniques.

Azobenzene photoisomerization probes cell membrane viscosity.

The viscosity of cell membranes is a crucial parameter that affects the diffusion of small molecules both across and within the lipid membrane and that is related to several diseases. Therefore, the possibility to measure quantitatively membrane viscosity on the nanoscale is of great interest. Here, we report a complete investigation of the photophysics of an amphiphilic membrane-targeted azobenzene (ZIAPIN2) and we propose its use as a viscosity probe for cell membranes. We exploit ZIAPIN2 trans-cis photoisomerization to develop a molecular viscometer and to assess the viscosity of Escherichia coli bacteria membranes employing time-resolved fluorescence spectroscopy. Fluorescence lifetime measurements of ZIAPIN2 in E. coli bacteria suspensions correctly indicate that the membrane viscosity decreases as the temperature of the sample increases. Given the non-homogeneity and the anisotropy of cell membranes, as supported by the photophysical characterization of the probe within the lipid bilayer, we shed new light on the intricate membrane rheology.

The Effect of an Intramembrane Light-Actuator on the Dynamics of Phospholipids in Model Membranes and Intact Cells.

The noncovalent intercalation of amphiphilic molecules in the lipid membrane can be exploited to modulate efficiently the physical status of the membrane. Such effects are largely employed in a range of applications, spanning from drug-delivery to therapeutics. In this context, we have very recently developed an intramembrane photo-actuator consisting of an amphiphilic azobenzene molecule, namely ZIAPIN2. The selective photo-isomerization occurring in the lipid bilayer induces a photo-triggered change in the membrane thickness and capacitance, eventually permitting to evoke light-induced neuronal firing both in vitro and in vivo. Here, we present a study on the dynamical perturbation in the lipid membrane caused by ZIAPIN2 and its vehicle solvent, dimethyl sulfoxide. Effects on the dynamics occurring in the picosecond time range and at the molecular level are probed using quasi-elastic neutron scattering. By coupling experiments carried out both on model membranes and intact cells, we found that DMSO leads to a general retardation of the dynamics within a more dynamically ordered landscape, a result that we attribute to the dehydration at the interface. On the other hand, ZIAPIN2 partitioning produces a general softening of the bilayer owing to its interaction with the lipids. These data are in agreement with our recent studies, which indicate that the efficacy of ZIAPIN2 in triggering cellular signalling stems from its ability to mechanically perturb the bilayer as a whole, by forming light-sensitive membrane spanning dimers.

Light-induced dipole moment modulation in diarylethenes: a fundamental study.

The dipole moment of photochromic diarylethenes is determined in solution for both the coloured and uncoloured forms by measuring the capacitance of a capacitor filled with a photochromic solution as a dielectric material. Diarylethenes with different substituents are investigated and the modulation of the dipole moment is related to their chemical structures. We determine a modulation of the dipole moment up to 4 Debye. We discuss the model used to obtain the dipole moment from the capacitance measurements and we compare the experimental results with the outcomes from DFT calculations. The results highlight the importance of conformational effects in the description of the dipole moment of diarylethenes.

Conducting Electrospun Poly(3-hexylthiophene-2,5-diyl) Nanofibers: New Strategies for Effective Chemical Doping and its Assessment Using Infrared Spectroscopy.

Vibrational spectroscopy allows the investigation of structural properties of pristine and doped poly(3-hexylthiophene-2,5-diyl) (P3HT) in highly anisotropic materials, such as electrospun micro- and nanofibers. Here, we compare several approaches for doping P3HT fibers. We have selected two different electron acceptor molecules as dopants, namely iodine and 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4TCNQ). In the case of iodine, we have explored the doping of the fibers according to several different procedures, i.e., by sequential doping both in vapors and in solution, and with a novel promising one-step method, which exploits the mixing of the dopant to the electrospinning feed solution. Polarized infrared (IR) spectroscopy experiments prove the orientation of P3HT chains, with the polymer backbone mainly running parallel to the fiber axis. After doping, P3HT fibers show very strong and polarized doping-induced IR active vibrations (IRAVs), which are the spectroscopic signature of the structure relaxation induced by the charged defects (polarons), thus providing an unambiguous proof of the effective doping. Raman spectroscopy complements the IR evidence: The Raman spectrum shows a clearly recognizable shift of the main band, the so-called effective conjugation coordinate band, in the doped samples. A simple protocol, which quantifies the evolution of the IRAV bands with time, allows monitoring of the doping stability over time and confirms that F4TCNQ is by far superior to iodine.

Modeling absorbance-modulation optical lithography in photochromic films.

A kinetic model describing the conversion of a photochromic layer under complex illumination conditions is applied to absorbance-modulation optical lithography to determine the influence of the material characteristics on the confinement to subdiffraction dimensions of the transmitted dose. We show that the most important parameters are the intensity ratio between the confining and writing beams, the overall absorption at the writing wavelength, the relative absorption coefficients, and the photoreaction quantum yields at the two wavelengths. As the confining beam ultimately determines the transferred dose pattern, we conclude that the modulation of the writing beam is not strictly necessary to produce subwavelength apertures.

Modulation of Mechanosensitive Potassium Channels by a Membrane-targeted Nongenetic Photoswitch.

Mechanosensitive ion channels are present in the plasma membranes of all cells. They play a fundamental role in converting mechanical stimuli into biochemical signals and are involved in several physiological processes such as touch sensation, hearing, and blood pressure regulation. This protein family includes TWIK-related arachidonic acid-stimulated K+ channel (TRAAK), which is specifically implicated in the maintenance of the resting membrane potential and in the regulation of a variety of important neurobiological functions. Dysregulation of these channels has been linked to various diseases, including blindness, epilepsy, cardiac arrhythmia, and chronic pain. For these reasons, mechanosensitive channels are targets for the treatment of several diseases. Here, we propose a new approach to investigate TRAAK ion channel modulation that is based on nongenetic photostimulation. We employed an amphiphilic azobenzene, named Ziapin2. In the dark, Ziapin2 preferentially dwells in the plasma membrane, causing a thinning of the membrane. Upon light irradiation, an isomerization occurs, breaking the dimers and inducing membrane relaxation. To study the effect of Ziapin2 on the mechanosensitive channels, we expressed human TRAAK (hTRAAK) channels in HEK293T cells. We observed that Ziapin2 insertion in the membrane is able per se to recruit hTRAAK, permitting the exit of K+ ions outside the cells with a consequent hyperpolarization of the cell membrane. During light stimulation, membrane relaxation induces hTRAAK closure, generating a consistent and compensatory depolarization. These results add information to the Ziapin2 mechanism and suggest that membrane deformation can be a tool for the nonselective modulation of mechanosensitive channels.

Membrane Targeted Azobenzene Drives Optical Modulation of Bacterial Membrane Potential.

Recent studies have shown that bacterial membrane potential is dynamic and plays signaling roles. Yet, little is still known about the mechanisms of membrane potential dynamics regulation-owing to a scarcity of appropriate research tools. Optical modulation of bacterial membrane potential could fill this gap and provide a new approach for studying and controlling bacterial physiology and electrical signaling. Here, the authors show that a membrane-targeted azobenzene (Ziapin2) can be used to photo-modulate the membrane potential in cells of the Gram-positive bacterium Bacillus subtilis. It is found that upon exposure to blue-green light (λ = 470 nm), isomerization of Ziapin2 in the bacteria membrane induces hyperpolarization of the potential. To investigate the origin of this phenomenon, ion-channel-deletion strains and ion channel blockers are examined. The authors found that in presence of the chloride channel blocker idanyloxyacetic acid-94 (IAA-94) or in absence of KtrAB potassium transporter, the hyperpolarization response is attenuated. These results reveal that the Ziapin2 isomerization can induce ion channel opening in the bacterial membrane and suggest that Ziapin2 can be used for studying and controlling bacterial electrical signaling. This new optical tool could contribute to better understand various microbial phenomena, such as biofilm electric signaling and antimicrobial resistance.

Electrospinning of pullulan-based orodispersible films containing sildenafil.

Feasibility of electrospinning in the manufacturing of sildenafil-containing orodispersible films (ODFs) intended to enhance oxygenation and to reduce pulmonary arterial pressure in pediatric patients was evaluated. Given the targeted subjects, the simplest and safest formulation was chosen, using water as the only solvent and pullulan, a natural polymer, as the sole fiber-forming agent. A systematic characterization in terms of shear and extensional viscosity as well as surface tension of solutions containing different amounts of pullulan and sildenafil was carried out. Accordingly, electrospinning parameters enabling the continuous production, at the highest possible rate, of defect-free fibers with uniform diameter in the nanometer range were assessed. Morphology, microstructure, drug content and relevant solid state as well as ability of the resulting non-woven films to interact with aqueous fluids were evaluated. To better define the role of the fibrous nanostructure on the performance of ODFs, analogous films were produced by spin- and blade-coating and tested. Interestingly, the disintegration process of electrospun products turned out to be the fastest (i.e. occurring within few s) and compliant with Ph. Eur. and USP limits, making relevant ODFs particularly promising for increasing sildenafil bioavailability, thus lowering its dosages.

Membrane Order Effect on the Photoresponse of an Organic Transducer.

Non-genetic photostimulation, which allows for control over cellular activity via the use of cell-targeting phototransducers, is widely used nowadays to study and modulate/restore biological functions. This approach relies on non-covalent interactions between the phototransducer and the cell membrane, thus implying that cell conditions and membrane status can dictate the effectiveness of the method. For instance, although immortalized cell lines are traditionally used in photostimulation experiments, it has been demonstrated that the number of passages they undergo is correlated to the worsening of cell conditions. In principle, this could impact cell responsivity against exogenous stressors, including photostimulation. However, these aspects have usually been neglected in previous experiments. In this work, we investigated whether cell passages could affect membrane properties (such as polarity and fluidity). We applied optical spectroscopy and electrophysiological measurements in two different biological models: (i) an epithelial immortalized cell line (HEK-293T cells) and (ii) liposomes. Different numbers of cell passages were compared to a different morphology in the liposome membrane. We demonstrated that cell membranes show a significant decrease in ordered domains upon increasing the passage number. Furthermore, we observed that cell responsivity against external stressors is markedly different between aged and non-aged cells. Firstly, we noted that the thermal-disordering effect that is usually observed in membranes is more evident in aged cells than in non-aged ones. We then set up a photostimulation experiment by using a membrane-targeted azobenzene as a phototransducer (Ziapin2). As an example of a functional consequence of such a condition, we showed that the rate of isomerization of an intramembrane molecular transducer is significantly impaired in aged cells. The reduction in the photoisomerization rate translates in cells with a sustained reduction of the Ziapin2-related hyperpolarization of the membrane potential and an overall increase in the molecule fluorescence. Overall, our results suggest that membrane stimulation strongly depends on membrane order, highlighting the importance of cell passage during the characterization of the stimulation tools. This study can shine light on the correlation between aging and the development of diseases driven by membrane degradation as well as on the different cell responsivities against external stressors, such as temperature and photostimulation.

Skeletal muscle cells opto-stimulation by intramembrane molecular transducers.

Optical stimulation and control of muscle cell contraction opens up a number of interesting applications in hybrid robotic and medicine. Here we show that recently designed molecular phototransducer can be used to stimulate C2C12 skeletal muscle cells, properly grown to exhibit collective behaviour. C2C12 is a skeletal muscle cell line that does not require animal sacrifice Furthermore, it is an ideal cell model for evaluating the phototransducer pacing ability due to its negligible spontaneous activity. We study the stimulation process and analyse the distribution of responses in multinuclear cells, in particular looking at the consistency between stimulus and contraction. Contractions are detected by using an imaging software for object recognition. We find a deterministic response to light stimuli, yet with a certain distribution of erratic behaviour that is quantified and correlated to light intensity or stimulation frequency. Finally, we compare our optical stimulation with electrical stimulation showing advantages of the optical approach, like the reduced cell stress.

Optical modulation of excitation-contraction coupling in human-induced pluripotent stem cell-derived cardiomyocytes.

Non-genetic photostimulation is a novel and rapidly growing multidisciplinary field that aims to induce light-sensitivity in living systems by exploiting exogeneous phototransducers. Here, we propose an intramembrane photoswitch, based on an azobenzene derivative (Ziapin2), for optical pacing of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The light-mediated stimulation process has been studied by applying several techniques to detect the effect on the cell properties. In particular, we recorded changes in membrane capacitance, in membrane potential (Vm), and modulation of intracellular Ca2+ dynamics. Finally, cell contractility was analyzed using a custom MATLAB algorithm. Photostimulation of intramembrane Ziapin2 causes a transient Vm hyperpolarization followed by a delayed depolarization and action potential firing. The observed initial electrical modulation nicely correlates with changes in Ca2+ dynamics and contraction rate. This work represents the proof of principle that Ziapin2 can modulate electrical activity and contractility in hiPSC-CMs, opening up a future development in cardiac physiology.

Tuning the quinoid versus biradicaloid character of thiophene-based heteroquaterphenoquinones by means of functional groups.

A series of quinoidal bithiophenes (QBTs) with controlled variations in steric hindrance and electron activity of the substituents has been synthesized. Evidence of their quinoidal versus biradicaloid ground-state electronic character has been experimentally detected and coherently identified as fingerprints by spectroscopic methods such as NMR, UV-vis, multiwavelength Raman. From this analysis, alkoxy groups have been shown to strongly affect the electronic structure and the ground-state energy and stability of QBTs. Quantum-chemical calculations correctly predict the experimental spectroscopic response, even while changing the alkyl on phenone from a tertiary carbon atom to secondary to primary toward an unsubstituted phenone, further confirming the validity of the approach proposed. A control of the electronic structure accompanied by negligible variations of the optical gap of the molecules has thus been demonstrated, extending the potential use of quinoidal species in fields ranging from photon harvesting to magnetic applications.

Conducting Electrospun Nanofibres: Monitoring of Iodine Doping of P3HT through Infrared (IRAV) and Raman (RaAV) Polaron Spectroscopic Features.

Aligned polymer nanofibres are prepared by means of the electrospinning of a chlorobenzene solution containing regioregular poly(3-hexyltiophene-2,5-diyl), P3HT, and poly(ethylene oxide), PEO. The PEO scaffold is easily dissolved with acetonitrile, leaving pure P3HT fibres, which do not show structural modification. Polymer fibres, either with or without the PEO supporting polymer, are effectively doped by exposure to iodine vapours. Doping is monitored following the changes in the doping-induced vibrational bands (IRAVs) observed in the infrared spectra and by means of Raman spectroscopy. Molecular orientation inside the fibres has been assessed by means of IR experiments in polarised light, clearly demonstrating that electrospinning induces the orientation of the polymer chains along the fibre axis as well as of the defects introduced by doping. This work illustrates a case study that contributes to the fundamental knowledge of the vibrational properties of the doping-induced defects-charged polarons-of P3HT. Moreover, it provides experimental protocols for a thorough spectroscopic characterisation of the P3HT nanofibres, and of doped conjugated polymers in general, opening the way for the control of the material structure when the doped polymer is confined in a one-dimensional architecture.

Morphology and Intramolecular Interactions in P(VDF-TrFE) Electrospun Nanofibers Doped with Disperse Orange 3 Dye: A Joint Infrared Spectroscopy and Electron Microscopy Study.

In this study, we describe a host-guest system consisting of a push-pull dye, the 4-amino-4'-nitroazobenzene (Disperse Orange 3, DO3), mixed with the copolymer poly(vinylidene fluoride-co-trifluoroethylene) [P(VDF-TrFE)] as a potential candidate for nonlinear optics (NLO) applications. We developed electrospun nanofibers of the polymer/dye blend, showing a highly anisotropic molecular structure, where DO3 molecules are mostly oriented parallel to the polymer chain, running in the fiber axis direction. The technique opens a way for obtaining non-centrosymmetric ordering of the NLO chromophore without requiring further poling. The supramolecular architecture is deeply investigated through infrared vibrational spectroscopy, which allows detecting a new phase involving DO3 molecules linked together by strong directional H-bonds. Electron microscopies highlight peculiar nanofiber morphologies with a preferred localization of DO3 at the surface layers.

Photochromic polyurethanes for rewritable CGHs in optical testing.

The development of photochromic Computer Generated Holograms (CGHs) to test any complex optics, such as aspheres and free-form optics, is described. A thermally irreversible photochromic polyurethane has been synthesized to give good thin films with a strong modulation of the optical transmission. The photochromic CGH has been tested with a simple interferometrical configuration showing promising results. The use of photochromic CGHs provides advantages over standard technologies, as rewritability and self developing.

Optical modulation of amplified emission in a polyfluorene-diarylethene blend.

A novel system for the modulation of amplified emission based on a polyfluorene/diarylethene (namely F8BT/DTP) blend is shown. The high sensitivity of amplified spontaneous emission (ASE) is exploited to achieve efficient emission modulation with a low-intensity control signal. Modulation is then characterized by photoluminescence (PL) lifetime measurements, photocurrent experiments, and density functional theory calculations. This system can also act as a photocurrent switch based on the same principle. This technique may represent a useful tool for fluorescence quenching and sensing as well as find application in organic photonics.

Kinetics of photochromic conversion at the solid state: quantum yield of dithienylethene-based films.

Quantum yield is one of the most important properties of photochromic systems. Unfortunately, a lack of data at the solid state exists, because measurements are intrinsically not straightforward. A kinetic model describing the conversion of the photoactive species is reported and both analytic and numeric solutions are provided according to relevant cases. The model is then applied to measure the quantum yield of dithienylethene-based polymers; the ring-opening quantum yield is measured for different laser beam profiles (i.e., Gaussian and uniform) and at different wavelengths, showing an increased value with increasing photon energy.

Biradicaloid character of thiophene-based heterophenoquinones: the role of electron-phonon coupling.

The quinoidal versus biradicaloid character of the ground state of a series of thiophene-based heterophenoquinones is investigated with quantum-chemical calculations. The role of the ground-state electronic character on molecular structure and vibrational properties is emphasized. The vibrational activities are experimentally determined and their analysis is performed by taking advantage of the definition of a collective vibrational coordinate (the R coordinate) maximizing the electron-phonon coupling, and connecting the quinoid and the aromatic biradicaloid resonance structures. The combined experimental and computational investigation supports the biradicaloid nature of the longer oligomers. The modulation of Raman intensities and frequency dispersion, experimentally observed by increasing the length of the chromophore, is shown to be reproduced well by model calculations on a single chromophore as a function of geometry displacements along the R-mode. These results underline the role of electron-phonon coupling in governing the structure-property relationship of highly conjugated organic compounds, underscoring the similarity of thiophene heterophenoquinone systems with other, more classical, oligophenylene and oligothiophene derivatives.

Microstructure and cytocompatibility of electrospun nanocomposites based on poly(epsilon-caprolactone) and carbon nanostructures.

Carbon nanostructures (CNSs) are attractive and promising nanomaterials for the next generation of tissue engineering scaffolds, especially in neural prosthesis. Optimizing scaffold vascularization may be an important strategy to promote the repair of damaged brain tissue. In this context, the idea was to evaluate the cell response of electrospun nanohybrid scaffolds loaded with CNSs. Fibrous composites based on poly(epsilon-caprolactone) (PCL) and CNSs were fabricated by means of electrospinning technique. High-purity carbon nanofibers (CNFs) and single-wall carbon nanotubes (SWNTs) were studied. A detailed microstructural characterization was performed to evaluate the most favorable experimental conditions for the realization of fibrous PCL/CNS fabrics. Electrospun mats comprised of rather uniform and homogeneous submicrometric fibers were obtained starting from 1:1 v/v mixture of tetrahydrofuran (THF) and N,N dimethylformamide (DMF). In vitro cytocompatibility tests were performed using rat cerebro-microvascular endothelial cells (CECs). Acquired results showed an increased cell viability for PCL/CNS nanocomposites, suggesting these materials as a suitable environment for endothelial cells. These results are indicative of the promising potential of CNS-based nanocomposites in biomedical devices for tissue engineering applications where endothelial functional properties are required.